Next Generation Sequencing Panels for Disorders of Sex Development Disorders of Sex Development – Overview Disorders of sex development (DSDs) occur when sex development does not follow the course of typical male or female patterning. Types of DSDs include congenital development of ambiguous genitalia, disjunction between the internal and external sex anatomy, incomplete development of the sex anatomy, and abnormalities of the development of gonads (such as ovotestes or streak ovaries) (1). Sex chromosome anomalies including Turner syndrome and Klinefelter syndrome as well as sex chromosome mosaicism are also considered to be DSDs. DSDs can be caused by a wide range of genetic abnormalities (2). Determining the etiology of a patient’s DSD can assist in deciding gender assignment, provide recurrence risk information for future pregnancies, and can identify potential health problems such as adrenal crisis or gonadoblastoma (1, 3). Sex chromosome aneuploidy and copy number variation are common genetic causes of DSDs. For this reason, chromosome analysis and/or microarray analysis typically should be the first genetic analysis in the case of a patient with ambiguous genitalia or other suspected disorder of sex development. Identifying whether a patient has a 46,XY or 46,XX karyotype can also be helpful in determining appropriate additional genetic testing. Abnormal/Ambiguous Genitalia Panel Our Abnormal/Ambiguous Genitalia Panel includes mutation analysis of 72 genes associated with both syndromic and non-syndromic DSDs. This comprehensive panel evaluates a broad range of genetic causes of ambiguous or abnormal genitalia, including conditions in which abnormal genitalia are the primary physical finding as well as syndromic conditions that involve abnormal genitalia in addition to other congenital anomalies. Our Abnormal/Ambiguous Genitalia Panel includes the genes listed below. Abnormal/Ambiguous Genitalia Panel AKR1C2 CREBBP DYNC2H1 HCCS LMNA PTPN11 SRD5A2 WT1 AR CYB5A ESCO2 HOXA13 MAP3K1 RIPK4 SRY ZFPM2 ARX CYP11A1 FAM58A HSD17B3 MKKS ROR2 STAR ATRX CYP11B1 FAT4 HSD3B2 MKS1 RSPO1 TBX15 B3GALTL CYP17A1 FEZF1 ICK NEK1 SALL1 TCTN3 BCOR CYP19A1 FIG4 IL17RD NR0B1 SCARF2 TSPYL1 BMP4 DHCR24 FRAS1 IRF6 NR5A1 SEMA3A UBR1 CDKN1C DHCR7 FREM2 KAL1 NSMF SETBP1 WDR60 CEP41 DHH GATA4 KISS1R OPHN1 SOX9 WNT4 CHD7 DNMT3B GRIP1 LHCGR POR SPECC1L WNT7A 46,XY Disorders of Sex Development/Complete Gonadal Dysgenesis Panel The 46,XY Disorders of Sex Development/Complete Gonadal Dysgenesis (46,XY DSD/CGD) Sequencing Panel includes sequence analysis of 26 genes associated with disorders of sex development (DSD) or complete gonadal dysgenesis (CGD) in patients with a 46,XY karyotype. Individuals with 46,XY CGD (also known as 46,XY sex reversal) have a 46,XY karyotype in conjunction with normal female external genitalia, “streak” gonads, absent sperm production, and presence of a uterus and fallopian tubes. 46,XY CGD has been associated with mutations or copy number variations in several genes, including SRY, DHH, NR5A1, and SOX9 (with campomelic dysplasia) (2). dnatesting.uchicago.edu • 773-834-0555 03/20 46,XY DSDs are characterized by one or more of the following in an individual with a 46,XY karyotype: ambiguous genitalia with mild to severe penoscrotal hypospadias, dysgenetic gonads, reduced/absent sperm production, and Müllerian structures that range from absent to presence of a uterus and fallopian tubes (4). Example of genes associated with 46,XY DSDs include AKR1C2, CYB5A, GATA4, and SRD5A2. In addition, a 46,XY DSD phenotype may be syndromic, as in Mowatt-Wilson syndrome (5), X-linked lissencephaly-2 (6), ATR-X syndrome (7), and Smith-Lemli-Opitz syndrome (8). Our 46,XY DSD/CGD Panel includes the genes listed below. 46,XY DSD/CGD Panel AKR1C2 CYB5A HCCS SOX9 AMH CYP11A1 HSD17B3 SRD5A2 AMHR2 CYP17A1 LHCGR SRY AR DHCR7 MAMLD1 WT1 ARX DHH MAP3K1 ZFPM2 ATRX DYNC2H1 NR5A1 B3GALTL GATA4 OPHN1 46,XX Disorders of Sex Development/Complete Gonadal Dysgenesis Panel The 46,XX Disorders of Sex Development/Complete Gonadal Dysgenesis (46,XX DSD/CGD) Panel includes 9 genes associated with disorders of sex development (DSD) or complete gonadal dysgenesis (CGD) in patients with a 46,XX karyotype. A range of phenotypes may be observed in patients with 46,XX DSD/CGD, from mullerian aplasia and hyperandrogenism (9) or ovarian dysgenesis (10), to adrenal hyperplasia with overvirilization. There are syndromic forms of 46,XX DSDs, including Peters-Plus syndrome (11) and WAGR syndrome (12). 46,XX complete gonadal dysgenesis (also referred to as 46,XX sex reversal, 46,XX true hermaphroditism, or ovotesticular DSD) may be observed in patients with presence of the SRY gene (13), in patients with copy number variations in SOX3 (14), or due to mutations in other genes, such as RSPO1 (15). Please note that due to the presence of a pseudogene, the CYP21A2 gene is not included on this panel. Our 46,XX DSD/CGD Panel includes the genes listed below. 46,XX DSD/CGD Panel B3GALTL HCCS RSPO1 CYP11B1 NR5A1 WNT4 CYP19A1 PSMC3IP WT1 Test metHods: Comprehensive sequence coverage of the coding regions and splice junctions of all genes in the panel is performed. Targets of interests are enriched and prepared for sequencing using the Agilent SureSelect system. Sequencing is performed using Illumina technology and reads are aligned to the reference sequence. Variants are identified and evaluated using a custom collection of bioinformatic tools and comprehensively interpreted by our team of directors and genetic counselors. All pathogenic and likely pathogenic variants are confirmed by Sanger sequencing. The technical sensitivity of this test is estimated to be >99% for single nucleotide changes and insertions and deletions of less than 20 bp. Our CNV detection algorithm was developed and its performance determined for the sole purpose of identifying deletions and duplications within the coding region of the gene(s) tested. Partial exonic copy number changes and rearrangements of less than 400 bp may not be detected by this methodology. Regions of high homology and repetitive regions may not be analyzed. This methodology will not detect low level mosaicism, balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype. The sensitivity of our deletion/duplication assay may be reduced when DNA extracted by an outside laboratory is provided. dnatesting.uchicago.edu • 773-834-0555 03/20 Abnormal or Ambiguous Genitalia Panel Sample specifications: 3 to10 cc of blood in a purple top (EDTA) tube Cost: $4000 CPT codes: See test page Turn-around time: 8 weeks Note: We cannot bill insurance for the above test 46,XY DSD/CGD Panel Sample specifications: 3 to10 cc of blood in a purple top (EDTA) tube Cost: $3000 CPT codes: See test page Turn-around time: 8 weeks Note: We cannot bill insurance for the above test. 46,XX DSD Panel Sample specifications: 3 to10 cc of blood in a purple top (EDTA) tube Cost: $2000 CPT codes: See test page Turn-around time: 8 weeks Note: We cannot bill insurance for the above test. Results: Results, along with an interpretive report, will be faxed to the referring physician. Additional reports will be provided as requested. All abnormal results will be reported by telephone. For more information about our testing options, please visit our website at dnatesting.uchicago.edu or contact us at 773-834-0555. References: 1. Clinical Guidelines for the Management of Disorders of Sex Development in Childhood. In: Development CotMoDoS, ed. Consortium on the Management of Disorders of Sex Development. Rohnert Park, CA: Intersex Society of North America, 2006. 2. Arboleda VA, Sandberg DE, Vilain E. DSDs: genetics, underlying pathologies and psychosexual differentiation. Nat Rev Endocrinol 2014: 10: 603-615. 3. Arboleda V, Vilain E. Disorders of Sex Development. In: Strauss III J, Barbieri R, eds. Yen & Jaffe's Reproductive Endocrinology: Saunders, 2009: 1367-1393. 4. Ostrer H. 46,XY Disorder of Sex Development and 46,XY Complete Gonadal Dysgenesis. In: Pagon R, Adam M, Ardinger H, eds. GeneReviews [Internet]. Seattle, WA: University of Washington, Seattle, 2008 [Updated 2009 Sep 15]. 5. Baetens D, Mladenov W, Delle Chiaie B et al. Extensive clinical, hormonal and genetic screening in a large consecutive series of 46,XY neonates and infants with atypical sexual development. Orphanet J Rare Dis 2014: 9: 209. 6. Kitamura K, Yanazawa M, Sugiyama N et al. Mutation of ARX causes abnormal development of forebrain and testes in mice and X-linked lissencephaly with abnormal genitalia in humans. Nat Genet 2002: 32: 359-369. 7. Gibbons RJ, Wada T, Fisher CA et al. Mutations in the chromatin-associated protein ATRX. Hum Mutat 2008: 29: 796-802. 8. Witsch-Baumgartner M, Fitzky BU, Ogorelkova M et al. Mutational spectrum in the Delta7-sterol reductase gene and genotype-phenotype correlation in 84 patients with Smith-Lemli-Opitz syndrome. Am J Hum Genet 2000: 66: 402-412. 9. Biason-Lauber A, Konrad D, Navratil F et al. A WNT4 mutation associated with Müllerian-duct regression and virilization in a 46,XX woman. N Engl J Med 2004: 351: 792-798. 10. Zangen D, Kaufman Y, Zeligson S et al. XX ovarian dysgenesis is caused by a PSMC3IP/HOP2 mutation that abolishes coactivation of estrogen-driven transcription. Am J Hum Genet 2011: 89: 572-579. 11. Weh E, Reis LM, Happ HC et al. Whole exome sequence analysis of Peters anomaly. Hum Genet 2014: 133: 1497-1511. dnatesting.uchicago.edu • 773-834-0555 03/20 12. Jadresic L, Leake J, Gordon I et al. Clinicopathologic review of twelve children with nephropathy, Wilms tumor, and genital abnormalities (Drash syndrome). J Pediatr 1990: 117: 717-725. 13. Nieto K, Peña R, Palma I et al. 45,X/47,XXX/47,XX, del(Y)(p?)/46,XX mosaicism causing true hermaphroditism. Am J Med Genet A 2004: 130A: 311-314. 14. Sutton E, Hughes J, White S et al.